Chemists, including organic, medicinal and natural product chemists, are routinely spending a large portion of their bench time for the separation of large and small quantities of organic mixtures to obtain pure compounds. Because of the time consuming nature of this process, there is a continued need in chemistry to separate organic mixtures more efficiently, rapidly and inexpensively. Liquid chromatography can provide access to pure compounds where distillation and/or recrystallization techniques may not. Methods of liquid chromatographic separation play an important role in the separation techniques available, as can be seen by the wide choices of liquid chromatographic products available to the chemists, and the demonstration of such products reported in scientific literature. These products can be categorized by those that allow the purification/separation of analytical quantities versus preparative quantities of organic material.
The problems associated with the efficient separation of small quantities of UV-active materials have largely been solved by Preparative Thin Layer Chromatography (PTLC) and Rotating Thin Layer Chromatography (RTLC). However, the separation of large quantities of organic materials relies on a number of additional column chromatographic techniques with distinct inefficiency.
Gravity column chromatography is one of the oldest liquid chromatographic techniques, time consuming and may require large volume of solvent for the separation of organic material. This process involves the elution of an organic mixture of compounds through an adsorbent, such as silica gel, alumina, or polysaccharide amongst others, by an appropriate eluant. The rate the organic material travels through the adsorbent is in part controlled by the composition of the eluant used as will as the unique interaction between solute and packing material. The collection of solute/eluant in small aliquots as they exit the column is followed by the identification of the contents of each receptacle typically by analytical Thin Layer Chromatography (TLC) and eventually consolidation of desired materials. This is the general basis of preparative liquid column chromatography.
Improvements on gravity column chromatography have centered around increasing resolution and shortening time spent on separation/purification of organic mixtures. G. A. Fisher and J. J. Kabara [Anal. Biochem. (1964), 9: 303-309] described a system of multi-bore columns for increased resolution during the separation of organic mixtures by liquid chromatography; W. B. Love [Chem. Ind. (London), (1967), 2062-2032] described the use of dry column chromatography by which compounds are separated with similar resolution to TLC; W. C. Still [J. Org. Chem. (1978), 43: 923-] reported a chromatographic method requiring less time, so-called Flash Liquid Chromatography (FLC) by which the eluant is propelled through the adsorbent via compressed gas air at a rate greater than that which could be provided by gravity, requiring shorter separation time with reasonable resolution; N. M. Targett [J. Org. Chem. (1979), 44: 4962-4964] reported enhanced resolution of flash chromatography by using fine particle of adsorbent (TLC grade) and so-called Vacuum Liquid Chromatography (VLC) by which a vacuum is utilized at the collection end of the column, drawing aliquots of eluant through the column at a rate greater than that provided by gravity, to successfully increase the efficiency of separation reducing solvent and time required for separation. Vacuum liquid chromatography (VLC) has been reported to possess all of the three desirable characteristics: efficiency, rapidity and low cost.
Other improvements include automated systems by which the eluant is automatically fractionated into receptacles to simplify the collection process but little improvement in the separation process. Additionally, instruments equipped with UV-detectors and/or fraction collectors for liquid chromatography and high performance liquid chromatography are commercially available, and can be used for monitoring and collecting large and small amounts of sample as it leaves the adsorbent in lieu of analytical TLC. Although this does afford chemist time, both of these type of instruments are expensive and the progress of the separation of organic mixture is still not monitored on the column during the separation, but rather is monitored at the collection stage of separation thus not allowing the chemist the capability to optimize separation efficiency. Therefore, failed separation must be rerun based upon the solvent profile used and resolution obtained. This requires chemist additional costs of time and material.
Investigation on improvements in "on column detection` of solutes has produced a number of patents. These improvements have been confined to the analytical and quantitative analysis of organic mixtures. For example, Gorman (U.S. Pat. No. 3,917,413) describes a rotating column, that quantifies the fractions of organic mixture on the column in a procedure unlike the densitometers or spectrophotometers used to measure emitted fluorescence radiation, reflected or transmitted light for the quantitative measurement of substances on thin layer chromatographic plates. The apparatus composed of measuring head, photomultiplier, optical bridge, monochromator and light source is not intended for the optimization of a separation profile. Sato (U.S. Pat. No. 4,774,686) describes a pyres column with a senin light source to determine more accurately the composition of organic mixture on the high performance liquid chromatographic column (HPLC). This prior art describes a useful column detection method specifically designed to use with expensive automated machinery and is not useful for preparative separation of organic mixtures.
To date all apparatuses of liquid column chromatographs for preparative scale separation of organic mixture lack a otrolumn monitoring system during the separation process and prior to collection so that the separation process of organic mixture can not be optimized.
The principal object of the present invention is to provide a set of novel apparatus and process for preparative separation of organic mixtures by flash and/or vacuum liquid chromatographs in particular when a quartz tubing is used as body of column and an adsorbent mixed with fluorescent material as the packing material. One can directly monitor the progress and efficiency of the separation of UV-active organic mixtures on the fluorescent adsorbent in the column by projecting UV-light onto it so as to optimized the separation process by adjusting the composition of eluant on time to ensure sufficient resolution and rapid elution. With the new apparatus of the present invention, the collection receptacles need only to be changed as desired, and the process or fraction collection thus is greatly simplified. Thus efficiency of separation process is greatly enhanced. Also, another object of the present invention is to provide a system for sample application simply by the compressed air.